Multiple myeloma (MM) is a hematologic malignancy characterized by proliferation of neoplastic plasma cells in the bone marrow. Although recent therapeutic options for MM have led to a considerable improvement of patient survival, the course of this disease remains lethal in most of cases. A wide number of complex genetic aberrations contributes to the multistep transformation process of plasma cells within the human bone marrow microenvironment (huBMM), which plays an essential role for growth, survival and the drug resistance of tumor cells. There is now a rising body of evidence demonstrating that these aberrations may affect the microRNAs (miRNA) expression in MM, which in turn translates into aberrant translation of messenger RNA. Therefore, the miRNA network is progressively disclosing its relevant key involvement in the pathogenesis of this important disease.
MicroRNAs are a class of regulatory non-coding RNAs of 19-25 nucleotides in length that act by targeting specific messenger RNAs (mRNAs) for degradation or inhibition of translation through base pairing to partially or fully complementary sites. At present, the miRNA network, which includes several hundreds of sequences, is involved in a variety of normal biological functions as well as in tumorigenic events, since deregulated miRNAs can act as oncogenes (Onco-miRNAs) or tumor-suppressors (TS-miRNAs). Changes in gene copy number, chromosomal translocation, mutations, transcriptional activation, epigenetic silencing and defective miRNA development are variably responsible of this deregulation. Therefore, miRNAs are emerging as new potential multi-target agents, due to their ability to target multiple genes, in the context of signaling networks involved in cancer promotion or repression.
Among miRNAs significantly deregulated in human cancer, miR-221 and miR-222 are of great interest for potential clinical applications. MiR-221 and miR-222 are highly homologous microRNAs encoded in tandem on the X chromosome, whose up-regulation has been recently described in several types of human tumors. MiR-221 and miR-222 act as oncogenic miRNAs that facilitate cell proliferation via down-regulation of p27 and/or p57, which negatively regulate the cell cycle progression from G1 to S phase.
Several reports suggested a key role of miR-221/222 in tumorigenesis. For example, Garofalo et al. have recently shown that up-regulation of miR-221/222 expression, by targeting PTEN and TIMP3, confers resistance to TRAIL-induced cell death and enhances tumorigenicity of lung and liver cancer cells. Chun-Zi et al. demonstrated that miR-221 and miR-222 by modulation of PTEN expression regulate radiosensitivity, cell growth and invasion of gastric cells. More recently, Pineau et al. showed that the treatment with LNA-modified miR-221 inhibitors reduced the growth of liver cancer cells that overexpressed miR-221/222 by targeting a DNA damage-inducible transcript 4 (DDIT4), a modulator of the mTOR pathway. In addition, Galardi et al. showed that the treatment with miR-221/222 antisense oligonucleotides reduces tumor growth by increasing intratumoral p27kip1 amounts.
Taken together, all these evidences support the notion that silencing miR-221/222 may represent a valuable anti-tumor option.
However, the variability of the molecular profile of human tumors does not allow transferring the experimental findings in a tumor system to further different tumor systems. Therefore, even if the above experiments and considerations may suggest the general idea of an anti-tumor activity of miR-221/222 inhibitors, said activity cannot be predicted or expected for tumors for which evidence has never been provided in widely recognized experimental models. Assessing the role of a miRNA in a tumor model requires a long and difficult scientific investigation based on the genetic and molecular mechanism of a specific tumor and this kind of investigation is also affected by dead end findings, thorough reconsideration and starting of new experimental designs.
WO2006108718 discloses the use of miRNA 221 and 222 for the treatment of GIST (gastrointestinal stromal tumor), kit-dependent acute leukemias, erythroleukemia, papillary thyroid carcinoma, or other kit-dependent tumors or disease conditions, and the use of inhibitors of said miRNAs in therapy for suppressed hematopoiesis in cancer patients, β-thalassemia, and other β-hemoglobin diseases. Multiple myeloma is not mentioned and it does not fall within any of the mentioned pathological conditions; in particular, it is not regarded as a c-kit-driven or c-kit-dependent tumor.
WO2011068546 discloses a diagnostic or prognostic indicator of multiple myeloma that comprises a global expression profile of total miRNAs in the subject with a pattern of 39 up-regulated and 1 down-regulated gene. MiRNA 222 is one of the 39 up-regulated miRNAs. However, the document only refers to a diagnostic or prognostic use of the miRNA pattern, no mention is made to the use of the individual miRNAs or their inhibitors in the treatment of multiple myeloma. In fact, a method for treating a subject having multiple myeloma is suggested but it relies on the administration to the patient of one or more therapeutic compounds that inhibits miRNA maturation pathway, in particular by inhibiting expression of one or both of AGO2 or DICER1 genes at the nucleic acid or protein level, by means of a shRNA, an antibody or other small molecule inhibitor. Therefore, it does refer to a whole miRNA blockade but does not refer to the inhibition of an individual miRNA among the 39 upregulated miRNAs. This approach produces a completely different profile of toxicity, which cannot be correlated with or compared to the profile obtainable with individual miRNA inhibition. The diagnostic method also relates to the global expression profile and not to the analysis of individual miRNAs. In table 5, expression of individual miRNAs is studied and associated with GEP-defined risk score and proliferation index. However, specific target are not identified and, in any case, mir-222 is not mentioned at all in the table. Besides, it is suggested that some individual miRNAs alone cannot significantly contribute to disease progression, while only their synergy might significantly contribute to MM disease progression. Therefore, there is no suggestion that the targeting of individual miRNA could be effective. Furthermore, in the experiment showing that higher total expression levels of some miRNAs might be associated with multiple myeloma disease initiation, the miRNAs used in the experiment were randomly selected; the identification of target miRNAs would therefore have required an undue burden of experimentation for the skilled in the art.
WO2012080721 discloses novel biological markers for plasma cell disorders, such as multiple myeloma, and in particular, it relates to the use of microRNAs, as diagnostic and prognostic markers in assays for detecting such disorders. Among the disclosed miRNAs, miR-221 is also disclosed but miR-16 is presented as the most significant since it is particularly highly expressed. As for the above document, no mention is made to the possible use of the disclosed miRNAs as a target in the treatment of said disorders. In fact, once a diagnosis of myeloma is made by detecting a set of miRNAs, the patients will undergo conventional therapy based on cyclophosphamide, thalidomide and dexamethasone.
US20100298410 discloses oligonucleotides, which target and inhibit in vivo certain microRNAs, among which miR-221 and miR-222 are also disclosed in a list. The document also generally provides for a method for the treatment of a disease associated with the presence or overexpression of the microRNAs. With regard to miR-221 and miR-222, the only therapeutic indications disclosed are prostate carcinoma, human thyroid papillary carcinoma and human hepatocellular carcinoma. Multiple myeloma is only mentioned in a list of cancers, which may be treated by the oligonucleotides of the invention. Said claimed oligomers are 7-10 nucleotides in length; in particular, the oligomer used for the inhibition of miR-221 and miR-222 is a 7-mer LNA-antimiR. Direct evidence of anti-myeloma activity of the disclosed oligomers is not provided in the application, and it cannot be inferred from experimental evidences of anti-tumor activity in other cancer systems, as already explained above.
The role of miR-221/222 in myeloma has been mentioned in the paper of Lionetti et al. (2009) (7), where the expression profiles of a series of miRNAs in a set of 38 MM patients have been studied. Said MM patients have been classified according to the TC (Translocation/Cyclin) classification which is based on the presence of the recurrent IGH (immunoglobulin heavy chain) chromosomal translocations and cyclins D expression: TC1: patients characterized by the t(11;14) or t(6;14) translocation; TC2: patients showing low to moderate levels of the CCND1 gene in the absence of any primary IGH translocation; TC3: patients that do not fall into any of the other groups; TC4: patients showing high CCND2 levels and the presence of the t(4;14) translocation; TC5: patients with either the t(14;16) or the t(14;20) translocation (Hideshima et al., Blood 2004). MiR-221/222 have been found to be deregulated in MM with significant up-regulation in a subset of patients classified as TC2, showing low to moderate levels of the CCND1 gene in the absence of any primary IGH translocation, and TC4, that shows t(4;14) translocation. However, a potential anti-tumor effect of any in vitro or in vivo perturbation of miR-221/222 is not addressed nor even mentioned in this document.
Therefore, there is still the need of an effective tool for the therapy of multiple myeloma.
In particular, multiple myeloma is a general disease comprising different disease subsets. This represents a relevant issue for the efficacy of treatment. In fact, different subtypes of patients can be identified, as according to the TC classification above disclosed, and different treatments may therefore be required. The specific target of patients with higher potential of benefit from miRNA inhibitor treatment would thus represent an extremely important advantage since it would avoid potential toxicity for non-responding patients, as well as costs for ineffective treatment. Within the general problem of the lack of an efficient tool for therapy of multiple myeloma, a more specific problem of efficiently targeting specific subset of patients can therefore be identified.
Another problem is the provision of an inhibitor of miR-221 and miR-222 able of inhibiting the two of them at the same time, thus providing an immediate therapeutical advantage.
Still another problem is to provide selective miRNA inhibitors.
A further problem is to provide inhibitors stable when administered in a subject.
It has now been found that silencing miR-221 and miR-222 with anti-miR-221 and/or anti-miR-222 exerts a powerful anti-tumor activity in vitro in MM cells and mostly evident in clinically relevant xenograft models of human MM. These models are recognized for starting a clinical development for the therapy of multiple myeloma.
Also, since miR-221 and miR-222 are particularly overexpressed in some MM subtypes (TC2 and TC4) their specific targeting represents a strong tool for rational patient selection and treatment.
The inhibition of miR transcriptional regulation requires the overcoming of several challenges.
In particular, an efficient miR inhibitor should have high binding affinity and nuclease resistance and be efficient for in vivo delivery. Different chemically modified oligonucleotides including 2′-O-methyl, locked nucleic acid (LNA) or 3′ cholesterol-conjugated with full o partial phosphorothioate backbone, have been proposed to silence miRs in vivo. In addition, tiny LNAs, fully LNA-modified phosphorothioate oligonucleotides, designed with perfect complementary miRs seed sequence, have been disclosed as valid miRs knockdown in different tumor model in vivo.
LNA miRNA-inhibitors commercially available have been used for the purposes of the present invention.
Further, novel miR-221 and miR-222 inhibitors (LNA-i-miR-221 and LNA-i-miR-222) able to antagonize miR-221 and miR-222 activity in vitro and in vivo have been designed in order to provide a more effective tool for the inhibition of said miRNAs.
Advantageously, these novel inhibitors are endowed with enhanced stability, allowing a better therapeutical efficacy.